Cardiovascular disease (CVD) is a major public health concern. Despite major progress in diagnosis and treatment over the years, CVD continues to represent the most frequent cause of morbidity and mortality worldwide. Platelets, which are central to normal hemostasis and limits blood loss after injury, are also the key players in pathological conditions, such as deep vein thrombosis and arterial thrombosis. They preserve vascular integrity and thereby prevent haemorrhage after injury. However, vascular damage, such as rupture of an atherosclerotic plaque, results in a platelet-dependent thrombus formation, which may lead to vascular occlusion with resultant hypoxia and infarction of distal tissues. The resulting clinical scenarios encompass stable and unstable angina, acute myocardial infarction (MI), ischaemic stroke and peripheral arterial occlusive disease.
The regulation of platelet-endothelial interaction, and thereby haemostasis and thrombosis, occurs due to precarious balance between activatory and inhibitory mechanisms that control platelet activation upon exposure to damaged tissues, yet enable platelets to remain quiescent in the undamaged circulation. In healthy vasculature, circulating platelets are maintained in an inactive state by nitric oxide and prostacyclin released by endothelial cells lining the blood vessels. Endothelial cells also express ADPase (adenosine diphosphatase), which degrades ADP released from red blood cells and activated platelets, thereby preventing further activation of platelets. When the vessel wall is damaged, the release of these endogenous anti-platelet substances is impaired and subendothelial matrix is exposed. Platelets adhere to exposed collagen and von Willebrand factor (vWF) through receptors that are constitutively expressed on the platelet surface. Adherent platelets integrate signals from the binding interaction, change their shape, secrete ADP from their dense granules, and synthesize and release thromboxane A2 (TXA2). The ADP and TXA2 that are released serve as platelet agonists by activating ambient platelets and recruiting them to the site of vascular injury. Disruption of the vessel wall also exposes platelets to collagen and tissue factor-expressing cells that further trigger the procoagulant response. In an advanced stage of activation, platelets stimulate blood coagulation by providing a surface at which the coagulation factors are activated to generate thrombin. In addition to converting fibrinogen to fibrin, thrombin also serves as a potent platelet agonist and recruits more platelets to the site of vascular injury. Activated platelets potentiate coagulation by expressing phosphatidylserine on their surface. When platelets are activated, glycoprotein (GP) IIb/IIIa (αIIbβ3), the most abundant receptor on the platelet surface, undergoes a conformational change, which increases its capacity to bind fibrinogen. Divalent fibrinogen molecules bridge adjacent platelets together to form platelet aggregates. Fibrin strands, generated by the action of thrombin, then weave these aggregates together to form a platelet-fibrin mesh.
The notion that the targeting of platelet function may be beneficial in the prevention of thrombosis is borne out by several clinical trials and the wide use of anti-platelet therapies. Anti-platelet agents can be sub classified, on the basis of their site of action, into those that inhibit (i) adhesion, (ii) activation, (iii) aggregation, or (iv) platelet-mediated links with inflammation. Of the currently available agents, aspirin, clopidogrel, dipyridamole, and cilostazol inhibit platelet activation, albeit via different mechanisms, whereas GPIIb/IIIa antagonists block platelet aggregation. However, there remains a substantial incidence of arterial thrombosis in patients on currently available anti-platelet therapy. Limitations of current therapies include weak inhibition of platelet function (for example, by aspirin), blockade of only one pathway of ADP mediated signalling (for example, by clopidogrel), slow onset of action (for example, of clopidogrel), interpatient response variability with poor inhibition of platelet response in some patients (for example, to aspirin and clopidogrel), the inability to transform the success of intravenous integrin αIIbβ3 antagonist therapy into oral therapy and the inability to completely separate a reduction in thrombotic events from an increase in bleeding events. Therefore, the successes and limitations of current therapies coupled with the advances made in our understanding of platelet biology are instructive in the design of new drugs to more effectively regulate validated targets.
Despite the complexity of extracellular matrix, platelet collagen interactions play a pivotal role in the initiation of hemostasis and thrombosis in vivo. As stable platelet adhesion and subsequent activation by collagen is mediated by platelet collagen receptors, the consequence of inhibition of either of these has gained considerable attention. Furthermore, human studies involving patients lacking either receptor, or equivalent mouse models, reveal reduced platelet responsiveness to collagen, with only mild deficiencies in haemostasis and do not lead to an increase in spontaneous bleeding tendency.
Thus, inhibition of platelet collagen-receptor activation may represent a novel pharmacological target in the search of more selective and specific antithrombotic agents for the prevention and/or treatment of acute occlusive arterial thrombosis, eg, myocardial infarction or stroke. Platelet interaction is the first step in the haemostatic process, where, extracellular matrix (ECM) is exposed at sites of injury. Among the macromolecular constituents of the ECM, collagen is considered to play a major role in this process, as in vitro it not only supports platelet adhesion through direct and indirect pathways but it also directly activates the cells in initiating aggregation and coagulant activity. The primary targets of existing anti-platelet therapy are molecules involved in platelet activation and aggregation. At present, there are no drugs in clinical use that block the initial tethering and adhesion of platelets to collagen and von Willebrand factor and hence their arrest on the blood vessel wall. The inhibition of this early step in thrombus formation is more likely to reduce or prevent the incidence of arterial thrombosis in patients of cardiovascular diseases [Review of the subject: PNAS, 2009 vol. 106 no. 3 719-724, Small-molecule inhibitors of integrin α2β1 that prevent pathological thrombus formation via an allosteric mechanism]. 
There have been several reports of peptide & proteins which inhibit platelet aggregation by inhibiting platelet activation by collagen & other endothelial derived activating molecules [FEBS Journal 277 (2010) 413-427, Aegyptin displays high-affinity for the von Willebrand factor binding site (RGQOGVMGF) in collagen and inhibits carotid thrombus formation in vivo; J Thromb Thrombolysis (2007) 24:275-282, Inhibition of collagen, and thrombin-induced platelet aggregation by Lansberg's hognose pit viper (Porthidium lansbergii hutmanni) venom; Acta Biochimica Polonica, Vol. 50 No. 4/2003, 1119-1128, Inhibition of collagen-induced platelet reactivity by DGEA peptide; FEBS Journal 273 (2006) 2955-2962, Identification and characterization of a collagen-induced platelet aggregation inhibitor, triplatin, from salivary glands of the assassin bug, Triatoma infestans; U.S. Pat. No. 5,851,839: Platelet aggregation inhibitors; 6 U.S. Pat. No. 5,756,454, Collagen-induced platelet aggregation inhibitor; U.S. Pat. No. 5,710,131, Inhibitor of collagen-stimulated platelet aggregation; U.S. Pat. No. 5,587,360, Platelet adhesion inhibitor].
However there are very few reports of small molecules which are capable of blocking platelet activation/aggregation by collagen & other endothelial derived activating molecules [(WO/1995/006038) Platelet aggregation inhibitors and references cited therein; U.S. Pat. No. 6,221,357, Flavonoids derived from citrus peels as collagen-induced platelet aggregation inhibitor; U.S. Pat. No. 5,814,636, Compounds with platelet aggregation inhibitor activity; U.S. Pat. No. 5,719,145, Amidine derivatives and platelet aggregation inhibitor containing the same; U.S. Pat. No. 5,698,692, Compound with platelet aggregation inhibitor activity; U.S. Pat. No. 5,629,321, Bicyclic compound and platelet aggregation inhibitor containing the same; J. Med. Chem. 2007, 50, 5457-5462, Small Molecule Inhibitors of Integrin α2β1; PNAS, 2009 vol. 106 no. 3 719-724, Small-molecule inhibitors of integrin α2β1 that prevent pathological thrombus formation via an allosteric mechanism; Cardiovascular Research 75 (2007) 782-792, Characterization of a novel and potent collagen antagonist, caffeic acid phenethyl ester, in human platelets: In vitro and in vivo studies; Arterioscler Thromb Vasc Biol 2007; 27; 1184-1190; EXP3179 Inhibits Collagen-Dependent Platelet Activation via Glycoprotein Receptor-VI Independent of AT1-Receptor Antagonism Potential Impact on Atherothrombosis; Phytother. Res. 16, 398-399 (2002), Human Platelet Aggregation Inhibitors from Thyme (Thymus vulgaris L.); J. Med. Chem., 2010, 53 (5), pp 2087-2093, Structural Basis for Platelet Antiaggregation by Angiotensin II Type 1 Receptor Antagonist Losartan (DuP-753) via Glycoprotein VI]
Amides of N-substituted pyroglutamic acids have been reported as moderate inhibitor of thrombin (Dikshit et al., 2001 Indian Patent 1206/DEL/2001) and has shown anti-thrombotic activity in mice model of thrombosis.
Therefore, the present invention focuses on the identification and preparation of pure diastereomers of a class of compounds which specifically inhibits collagen mediated platelet aggregation without affecting other regulatory and physiologically relevant platelet functions, and thus provides a clinically useful anti-thrombotic agent. The present invention makes pure diastereomers for improvement of their activity compared to the mixture of diastereomers.